J. Hescheler

Inward-rectifying channels in isolated patches of the heart cell membrane: ATP-dependence and comparison with cell-attached patches

Inward rectifying potassium single-channel currents were studied in the membrane of guinea pig cardiac myocytes. In isolated inside-out patches two different channels were observed: a channel of 25 pS conductance ([K+]o=147 mM,T=21° C), if the solution at the cytoplasmic face of the patch contained 4 mM ATP and a channel of 80 pS conductance without ATP. The 25-pS-channel was also regularly seen in cell-attached patches (Sakmann and Trube 1984a, b), but the 80-pS-channel appeared only after inhibiting cellular metabolism by DNP. The percentage of time which the 25-pS-channel spent in the open state was 3.3 times larger in isolated patches compared to cell-attached patches. However, both types of single channel currents disappeared several minutes after the isolation of the patches.In contrast to the 25-pS-channel, the 80-pS-channel, which is activated by the lack of ATP, carried measurable outward currents saturating at 1.5 pA (inward rectification). It is suggested that the 80-pS-channel mediates part of the increase in potassium current during metabolic inhibition. The openings of this channel appeared in bursts. The mean open time was 1.6 ms and the mean duration of the gaps within bursts 0.33 ms at −80 mV.

On the mechanism of muscarinic inhibition of the cardiac Ca current.

The mechanism of muscarinic inhibition of the Ca-current (ICa) was studied in ventricular myocytes of guinea pig hearts and the following results were obtained. 1. Acetylcholine (ACh) in concentrations up to 10−4 M had little effect, if any, onICa in control cells. 2. ACh reduced the isoprenaline (ISP)-induced increase ofICa. The doseresponse-relation (ISP concentration vs.ICa density) was shifted by ACh towards higher ISP concentrations. But both, at low and high ISP concentrations ACh had nor or little effect. 3. ACh was ineffective whenICa was increased by dialysing the cell with catalytic subunit of cAMP-dependent protein kinase or cAMP. 4. ACh reducedICa enhanced by isobutylmethylxanthine or by forskolin. 5. ACh did not depressICa when the cell was dialysed with the nonhydrolysable GTP-derivative, GMP-PNP. In this condition the β-adrenergic enhancement ofICa was also absent. 6. Pertussis toxin, which is known to inhibit the inhibitory transducerprotein (Ni), abolished the ACh response.We concluded from these results that ACh depressesICa by inhibiting, via Ni, the cAMP production.

Does the organic calcium channel blocker D600 act from inside or outside on the cardiac cell membrane

AbstractThe effects of extra- and intracellularly applied D600 (methoxyverapamil) and D890 (a quarternary derivative) on the action potentials of isolated guinea pig myocytes were compared. We also studied the extracellular effects of these drugs on the calcium current (hybride sucrose gap) and contractile force of right ventricular trabeculae of the cat heart.The following results were obtained:1.In ventricular trabeculae D600 suppressed the calcium current, tension and the plateau of the action potential. In contrast, D890 even in a 50 times higher concentration did not display any effect on these parameters.2.In single isolated cells external application of D890 did not alter the configuration of the action potential. In contrast, external application of D600 suppressed the plateau and shortened the action potential in a dose-dependent way.3.Intracellular injection of D600 or D890 strongly lowered the height of the plateau and abbreviated the action potential. The onset of the effects of both drugs was more rapid on intracellular application than that of external D600. Whereas the effect of an inracellular injection of D600 was reversible, that of D890 was not. These results support the hypothesis that the organic calcium channel blocker D600 enters the cell in the uncharged lipid soluble form and reaches its receptor associated with the calcium channel from inside. Because of its inability to pass the hydrophobic cell membrane, D890 is ineffective from outside but displays blocking effects on intracellular application.

“Run-down” of the Ca current during long whole-cell recordings in guinea pig heart cells: role of phosphorylation and intracellular calcium

We examined by a statistical approach the decrease of the Ca current (“run-down”) during long-lasting recordings with the whole-cell patch-clamp technique in guinea pig ventricular myocytes. The results are as follows. (1) Run-down of the Ca current (ICa) occurs in three phases (T1–T3). T1 (38±19 min,n=135) and T3 (35±17 min,n=23) are characterized by a slow rate of decay ofICa [90±20 and 60±20 nA·cm−2·min−1, respectively]. T1 and T3 are separated by T2 (6±4 min,n=135) during which the current decays quickly [1200±230 nA·cm−2·min−1]. Between the onsets of T1 and T3,ICa decreases from 11±3 to 3.5±1 μA/cm2. (2) Normalized current-voltage relationship, reversal potential and voltage-dependencies of steady-state activation and inactivation ofICa are globally shifted toward more negative potentials during the run-down process by 10–15 mV. (3)ICa3 measured during T3 retains the pharmacological properties (blockade by D600, NiCl2 and CoCl3, increase by isoprenaline and insensitivity to tetrodotoxin) of the originalICa. (4) Intracellular perfusion of the nonhydrolysable ATP analogue AMP-PNP does not prevent the occurrence of T2, suggesting that a phosphorylation-dephosphorylation process is not involved in the fast run-down ofICa. (5) With 0.1 mM EGTA in the pipette, addition of 3 mM ATP significantly prolongsICa survival. No improvements are obtained by increasing the ATP concentration to 10 mM or replacing ATP with creatine phosphate. With 3 mM ATP present, increasing the EGTA concentration to 10–20 mM doublesICa survival time. EGTA alone (10 mM) is less effective than the mixture 3 mM ATP-0.1 mM·EGTA. Intracellular perfusion with a cytoplasmic extract considerably prolongs T2 and the overallICa survival. (6) The results are consistent with the hypothesis that run-down ofICa can partially be explained by a rise in intracellular Ca concentration and a loss of high energy compounds. Beneficial effect of ATP might include an increased capability of the cells to either extrude or sequester intracellular Ca, and a protection against enzymatic proteolysis.

Modulation of Ca current during the phosphorylation cycle in the guinea pig heart

The calcium current (ICa) in the heart is increased by phosphorylation of a protein which is part of, or close to, the Ca channel. The phosphorylation is catalysed by cAMP-dependent protein kinase (cAMP-PK). The question whether dephosphorylated channels are available to open on depolarization was examined in ventricular myocytes of guinea pig by recording whole cellICa during dialysis with either regulatory (R) subunit of cAMP-PK or protein kinase inhibitor (PKI) or adenosine-5′-(γ-thio)-triphosphate (ATPγS). The following results were obtained: 1) R subunit reduced and PKI reversed the isoprenaline (ISP)-induced enhancement ofICa, suggesting their ability to inhibit cAMP-PK. 2) R subunit and PKI, however, reduced basal (i.e. non β-adrenergically stimulated)ICa only by about 20%. 3) Dialysis with ATPγS resulted in a slow increase in basalICa, presumably due to dephosphorylation-resistant thiophosphorylation. 4) When, however, the cell was dialyzed with PKI the effect of ATPγS was almost completely suppressed, suggesting no detectable phosphorylation related to the channel activity in this condition. These results support the view that even in the dephosphorylated state Ca channels are available to open on depolarization and that phosphorylation by cAMP-PK increases the opening probability.

Effects of a protein phosphatase inhibitor, okadaic acid, on membrane currents of isolated guinea-pig cardiac myocytes

The effects of a protein phosphatase inhibitor, okadaic acid (OA), were studied on membrane currents of isolated myocytes from guinea-pig cardiac ventricle. The whole-cell Ca2+ current (ICa) was recorded as peak inward current in response to test pulse to O mV. Extracellular application of OA (5–100μM) produced an increase ofICa. The effect was markedly enhanced when the myocyte was pretreated with threshold concentrations of isoprenaline.ICa was increased from 11.3±0.8μA cm−2 to 19.0±1.1μA cm−2 (n=4) by 5μM-OA in the presence of 1nM-isoprenaline. The delayed rectifier current was also slightly increased. Furthermore, the wash-out time of the β-adrenergic increase ofICa was markedly prolonged by OA. The β-adrenergic stimulation of cardiac Ca2+ current is thought to be mediated by cAMP-dependent phosphorylation. The present results strongly suggest that the effect of OA onICa is related to inhibition of endogenous protein phosphatase activity which is responsible for the dephosphorylation process. By the isotope method, the inhibitory effect of OA on different types of phosphatase was compared. OA had a relatively high specificity to type 1-, and type 2A-phosphatases.

Changes of membrane currents in cardiac cells induced by long whole-cell recordings and tolbutamide

Single isolated myocytes were obtained from the ventricles of adult guinea pig hearts. The whole-cell recording configuration of the patch-clamp technique was used to measure membrane currents. A decrease (run-down) of the Ca2+ inward current and an increase of a time-independent K+ outward current were observed during long lasting (1–3 h) recordings. The time at which the outward current developed depended on the intracellular ATP concentration in the pipette, suggesting that this current is identical to the ATP-dependent K+ current described by Noma and Shibasaki (1985). However, the maximum outward current reached in the experiments was independent of the ATP concentration indicating a limited diffusion of ATP in the cell interior. In single-channel experiments on isolated patches of cell membrane and in whole-cell recordings the ATP-dependent K+ current could be blocked by the hypoglycaemic sulphonylurea tolbutamide. The IC50 of 0.38 mM was about 50 times higher than that reported for pancreatic β-cells (Trube et al. 1986). The Ca2+ inward current and the inwardly retifying K+ current were not affected by tolbutamide (3 mM).

A possible physiological role of the Ca-dependent protease calpain and its inhibitor calpastatin on the Ca current in guinea pig myocytes

The decrease (‘run-down’) of the L-type Ca2+-current during long-lasting recordings with the whole-cell patch-clamp technique was examined in guinea pig ventricular myocytes. We have tested whether proteolysis is involved in the decay of the Ca2+-current by intracellular application of several concentrations of the Ca2+-dependent proteases calpain I and II, as well as their endogenous inhibitor calpastatin. The major finding was that calpain I and calpain II accelerated the Ca2+-current run-down in a concentration dependent manner, whereas calpastatin retarded it. These observations indicate that a proteolytic degradation of Ca2+-channels might be the reason for the run-down phenomenon.

Adrenoceptor‐mediated changes of excitation and contraction in ventricular heart muscle from guinea‐pigs and rabbits.

1. The influence of alpha‐adrenoceptor stimulation on mechanical and electrophysiological parameters was investigated in ventricular preparations from guinea‐pigs and rabbits. Action potential and force of contraction were measured in papillary muscles and ionic currents were measured in isolated myocytes. 2. The effects of alpha‐adrenoceptor stimulation were compared with those of beta‐adrenoceptor stimulation. 3. In the guinea‐pig the stimulation of alpha‐adrenoceptors caused a small increase in the force of contraction (less than 10% of the response to beta‐adrenoceptor stimulation) which was not accompanied by any increase of the slow calcium inward current. beta‐Adrenoceptor stimulation produced large increases in both force of contraction and slow inward calcium current. The noradrenaline‐induced increase in the slow inward calcium current was not affected by phentolamine. 4. In the rabbit, alpha‐adrenoceptor stimulation produced large increases in the force of contraction (about two thirds of those seen in response to beta‐adrenoceptor stimulation). Whereas beta‐adrenoceptor stimulation also produced large increases in both maximal upstroke velocity of slow‐response action potentials and slow inward calcium current, there was almost no change of both parameters in response to alpha‐adrenoceptor stimulation. 5. We conclude that, first, the contribution of alpha‐adrenoceptors to adrenoceptor‐mediated changes of force of contraction is minimal in the guinea‐pig ventricle, and second, the pronounced changes of force of contraction in the rabbit ventricle in response to alpha‐adrenoceptor stimulation are unrelated to changes in the slow inward calcium current.

Pain Perception in Mice Lacking the β3 Subunit of Voltage-activated Calcium Channels

The importance of voltage-activated calcium channels in pain processing has been suggested by the spinal antinociceptive action of blockers of N- and P/Q-type calcium channels as well as by gene targeting of the α1B subunit (N-type). The accessory β3 subunits of calcium channels are preferentially associated with the α1B subunit in neurones. Here we show that deletion of the β3 subunit by gene targeting affects strongly the pain processing of mutant mice. We pinpoint this defect in the pain-related behavior and ascending pain pathways of the spinal cordin vivo and at the level of calcium channel currents and proteins in single dorsal root ganglion neurones in vitro. The pain induced by chemical inflammation is preferentially damped by deletion of β3 subunits, whereas responses to acute thermal and mechanical harmful stimuli are reduced moderately or not at all, respectively. The defect results in a weak wind-up of spinal cord activity during intense afferent nerve stimulation. The molecular mechanism responsible for the phenotype was traced to low expression of N-type calcium channels (α1B) and functional alterations of calcium channel currents in neurones projecting to the spinal cord.